We have continued to develop selective biophysical measurement systems (biological atomic force microscopy (Bio-AFM) platforms, Quartz Crystal Microbalance-Dissipation (QCM-D), and optical microscopy and spectroscopy), and to apply these technologies to important biomedical investigations in collaboration with outstanding NIH intramural and extramural scientists. We are working toward broader and more insightful applications of multifunctional, multimodal, and multiplatform AFM imaging and single molecule force spectroscopy (SMFS) for cellular and macromolecular studies. On biomedical applications, we have continued our broad range of collaborations that include: (1) We have advanced our commitment to developing a better clinical vaccine toward enhanced immunological response and eventual eradication of malaria. Over several years and via Bio-AFM and related bioanalysis, we have investigated the macromolecular structure and nanomechanical properties of more and malaria vaccine candidates and virus-like-particle (VLP) or liposome carriers with Dr. David Narum (NIAID, NIH) and other collaborators. These malaria protein antigens and vaccine carriers are produced via recombinant-protein biotechnology, purified, and characterized in a manner suitable for human trials and scale-up production. Biophysical characterization at single macromolecule and assembly level using Bio-AFM imaging and force spectroscopy are helping to define these vaccine constructs along the developmental phases. In this year, we worked on extracting and characterizing Plasmodium falciparum lipid rafts and GPI-anchored proteins to improve mechanistic understanding of the malaria parasites and pathogen-host interactions. This work follows upon our published observation of a reversible conformation change and adhesion domain masking in the Plasmodium falciparum circumsporozoite protein (CSP), the leading malaria vaccine target. (2) Understanding and fighting cancer is in our focus this year. We have expanded our collaborations on multifunctional nanomedicine and theranostics with Dr. Xiaoyuan Chen (laboratory of Molecular Imaging and Nanomedicine, NIBIB) and co-investigators including Dr. Peng Huang (NIBIB) and Dr. Zhe Wang (NIBIB). We have contributed to several published studies such as using tumor-specific formation of enzyme-instructed supramolecular assemblies as cancer theranostics. We are examining critically broader applications of Bio-AFM and QCM-D methodology for investigating nanoparticle theranostics, characteristics of the cancer cells and stem cells, and biomedical systems related to targeted therapy and immunotherapy for advanced cancers. (3) We have continued our multi-year Bio-AFM studies of protein clathrin and assemblieswith collaborators including Prof. Eileen Lafer and Prof. Rui Sousa (Univ. Texas Health Sciences Center, San Antonio), Dr. Ralph Nossal (NICHD) and Dr. Dan Sackett (NICHD). Clathrin is a key protein for receptor-mediated endocytosis and intracellular trafficking. Further Bio-AFM and QCM-D measurements have been pursued this year to characterize clathrin and its assembled structures, as well as interaction with several partner proteins important to the function of cells, especailly. We contributed this year to a Nature Structure and Molecular Biology publication entitled Clathrin-coat disassembly illuminates the mechanisms of Hsp70 force generation. This collaboration has expanded toward bio-AFM studies of microtubules interacting with several approved or developing anti-cancer drugs. Also in the area of exocytosis and endocytosis, we contributed to a new Nature Communications paper with Dr. Ling-gang Wu (NINDS) and coworkers on cell membrane and intracellular structural changes to better understand synaptic transmission in brain. (4) We have collaborated further with Dr. Andrew Doyle and Dr. Kenneth Yamada (Laboratory of Cell and Developmental Biology, NIDCR) on nanomechanics and structural properties of reconstituted extra cellular matrix (ECM)-like collagen gels via Bio-AFM force spectroscopy to investigate cell adhesion, migration, and other dynamic behaviors. A paper is published in Nature Communications early this year. Collaborating also with Dr. Raimon Sunyer (Institute for Bioengineering of Catalonia, Spain), we refined force spectroscopy and quantitative nanomechnical mapping (QNM) approaches to explore more completely tissue-mimicking soft material and tissue specific extracellular matrices. (5) With Dr. Richard Hendler(NHLBI), Dr. Curtis Meuse(NIST) and others, we have advanced our Bio-AFM and biophysical studies of amyloid-beta fibrils in the Alzheimer's disease, especially to resolve time-resolved assembly pathways in physiologically relevant fluid and surface environments. We advanced on a project with Prof. Qi Lu (Delaware State University) and co-investigators, on the effect of nanoparticles on lipid domains and membrane properties. Finally, combining AFM and optical microscopy in other continuing and new collaborations, we have investigated protein assemblies, DNA crosslinking, DNA-protein interactions and cell cycles, critically important to disease mechanisms and bionanotechnology.